Agricultural microbial inoculant compositions and uses thereof

ABSTRACT

The present disclosure provides novel agricultural microbial inoculant compositions for uses in promoting plant growth, plant productivity and/or soil quality. The novel microbial inoculant compositions comprise one or more microbial species, one or more urease inhibitors and/or one or more nitrification inhibitors. The present disclosure also provides fertilizer compositions comprising said microbial inoculant compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/169,942, filed on Jun. 2, 2015, which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present disclosure relates to novel agricultural microbial inoculantcompositions for uses in promoting plant growth, plant productivityand/or soil quality. The novel microbial inoculant compositions compriseone or more microbial species, one or more urease inhibitors and/or oneor more nitrification inhibitors. The present disclosure also relates tofertilizer compositions comprising said microbial inoculantcompositions, formulations and the uses thereof.

BACKGROUND

The use of fertilizers to enhance plant and crop production and overcomepoor soil quality is widespread. Most commonly employed commerciallyavailable nitrogen containing fertilizers are inorganic chemicalfertilizers such as urea. The extended use of urea is often associatedwith negative environmental consequences, such as nitrate contaminationin run off and ground water, and emission of ammonia and nitrous oxideto the atmosphere. Attention to nitrogen fertilizer application hasshifted from the role of promoting crop production to alleviatingenvironmental pollution. There are a variety of new management practicesand technologies that can promote nitrogen use efficiency and alleviateenvironmental pollution.

One of the widely used technologies is the application of a ureaseinhibitor in combination with the urea treatment. The urea component offertilizer applied to the soil becomes a source of ammonia as a resultof urease catalyzed hydrolysis of urea, an enzyme produced by numerousfungi and bacteria that is well known to skilled artisans. Ureaseinhibitors can slow down the conversion rate of urea to ammonia, therebysignificantly reducing the quantity of urea that otherwise has to beapplied on the soil by reducing the amount of ammonia volatilization.One of the most common urease inhibitors is N-(n-butyl) thiophosphorictriamide (NBPT) (See e.g. U.S. Pat. No. 5,698,003).

Another widely used technology is the application of nitrificationinhibitors to significantly reduce nitrate leaching and gaseous nitrogenemissions. Most nitrogen supplied as a commercial fertilizer isultimately transformed to a nitrate form of nitrogen. In the presence ofadequate oxygen, warm temperatures, and some moisture, ammonium-N isconverted to nitrate-N through a biochemical process known asnitrification that requires two forms of soil bacteria. The firstbacterium Nitrosomonas converts ammonium-N to nitrite-N. The secondbacterium Nitrobacter converts nitrite-N to nitrate-N. Nitrificationinhibitors have one primary way of delaying the nitrification process byinhibiting the bacteria Nitrosomonas in the area where ammonium is to bepresent. Some widely used nitrification inhibitors that are commerciallyavailable include 2-chloro-6-(trichloromethyl)-pyridine (Nitrapyrin) anddicyandiamide (DCD).

In addition to the application of chemical enzyme inhibitors such asurease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) andnitrification inhibitors such as dicyandiamide (DCD), fertilizercompositions comprising microorganisms (so-called “bio-fertilizers” or“bio-stimulants”) are increasingly considered as alternatives toconventional chemical fertilizers. The ability of specific bacterialspecies to promote plant growth has long been recognized. For example,nitrogen-fixing bacteria such as Rhizobium species provide plants withessential nitrogenous compounds. Species of Azotobacter and Azospirillumhave also been shown to promote plant growth and increase crop yield,promoting the accumulation of nutrients in plants. However bacteria ofthese genera are often unable to compete effectively with native soiland plant flora, thereby requiring the application of impracticallylarge volumes of inocula.

SUMMARY OF THE INVENTION

To date, urease inhibitors and nitrification inhibitors have met withvaried success, while bio-fertilizers have typically met with limitedsuccess. Thus, there remains a need for improved fertilizers orfertilizer additives and methods that are effective in providingnutrients for plant growth and are environmentally safe andnon-hazardous. One solution is to provide a combination of ureaseinhibitors and/or nitrification inhibitors with bio-fertilizers.Nevertheless, the combination of urease inhibitors and/or nitrificationinhibitors with bio-fertilizers is not straight forward. First, ureaseinhibitors and/or nitrification inhibitors can weaken or kill thebio-fertilizers when combined. Second, urease inhibitors and/ornitrification inhibitors are typically dispensed in a solvent system(e.g. glycol, complex amines, aryl alcohols), which can also weaken orkill the bio-fertilizers.

WO 2015/104698 A2 disclosed combining a urease inhibitor with manymicrobial pesticides. However, it did not disclose any solvent systemthat could provide reasonable viability for the listed microbialpesticides. In addition, it did not identify any solvent-tolerantbacteria.

Solvent-tolerant bacteria are potentially useful in many applications ofmicrobial transformation for environmental remediation as well as inbiotechnological processes. Organic solvent tolerance may be aspecies-specific property and may not be easily predictable. See, forexample, Association of organic solvent tolerance and fluoroquinoloneresistance in clinical isolates of Escherichia coli. Anbu, P., Journalof Antimicrobial Chemotherapy, (1998) 41, 111-114. Most of the reportedand well-studied solvent-tolerant bacteria are Gram-negative bacteria.Gram-negative bacteria have the advantage of having an additional outermembrane that protects the cytoplasmic membrane by reducing theperiplasmic concentrations of harmful solvents to acceptable levels.Owing to the inherent disadvantage of lacking an outer membrane, only afew Gram-positive organisms have been reported to exhibit solventtolerance. See, for example, Isolation and characterization of a novelorganic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilicGram-positive bacterium. Gao, Y., Journal of Applied Microbiology, 110,472-478.

Surprisingly, in extensive efforts to identify solvent-tolerant bacteriato promote plant health, plant nutrition, and/or soil health, a fewagriculturally beneficial Gram-positive organisms are identified to beviable in some selected organic solvents while some Gram-negativeorganisms are not viable in the same organic solvents. For example,tested Gram-positive organisms species Bacillus amyloliquefaciens,Bacillus licheniformis, Bacillus thuringiensis and Bacillus pumilus havedemonstrated viability time range from at least 2 hours to at least 21days in a solution with at least one of the organic solvents propyleneglycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutylether, glycerol, and dimethyl sulfoxide (DMSO). However, none of thetested agriculturally beneficial Gram-negative species Pseudomonasfluorescens, Pseudomonas putida, and Pseudomonas chlororaphisdemonstrated viability in the tested organic solvents.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising at least one microbial strainfrom one or more microbial species, and at least one active agent,wherein the active agent is a urease inhibitor or a nitrificationinhibitor or a combination thereof, and further wherein the at least onemicrobial strain is present at an effective amount to promote planthealth, plant nutrition, and/or soil health in the presence of theactive agent.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof; and

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. a urease inhibitor selected from the group consisting ofN-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphorictriamide, thiophosphoryl triamide, phenyl phosphorodiamidate,N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide,phosphoric triamide, hydroquinone, p-benzoquinone,hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines,thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises a urease inhibitor and at least oneorganic solvent, wherein said urease inhibitor is selected from thegroup consisted of N-(n-butyl)thiophosphoric triamide (NBPT),N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenylphosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexylthiophosphoric triamide, phosphoric triamide, hydroquinone,p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines,thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof; andwherein said organic solvent is selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

Part B, wherein Part B comprises at least one Bacillus species selectedfrom the group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises at least one organic solvent selectedfrom the group consisting of propylene glycol (PG),N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether,glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and

Part B, wherein Part B comprises at least one Bacillus species selectedfrom the group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

In another embodiment, the present disclosure provides a method ofenhancing a yield trait in a subject plant as compared to the yieldtrait of a reference or control plant, the method comprising contactinga subject plant, plant part, plant seed, or surrounding soil with aneffective amount of a microbial inoculant composition of the presentdisclosure.

The urease inhibitor or nitrification inhibitor can mitigate nitratecontamination in run off and ground water, and the emission of a largeamount of ammonia and nitrous oxide to the atmosphere. The microbialspecies can further promote plant health, plant nutrition, and soilhealth. The combination of both chemical enzyme inhibitors and microbialspecies in suitable compositions and formulations may serve as a betterapproach to improve the efficiency of nitrogen-based fertilizer usage byimproving plant productivity, soil quality, and the overallenvironmental sustainability.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising at least one microbial strainfrom one or more microbial species, and at least one active agent,wherein the active agent is a urease inhibitor or a nitrificationinhibitor or a combination thereof, and further wherein the at least onemicrobial strain promotes plant health, plant nutrition, and/or soilhealth in the presence of the active agent.

In another embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising at least one microbial strainfrom one or more microbial species, and at least one active agent,wherein the active agent is a urease inhibitor or a nitrificationinhibitor or a combination thereof, further wherein the at least onemicrobial strain promotes plant health, plant nutrition, and/or soilhealth in the presence of the active agent, wherein one or moremicrobial species are selected from the following group:

(1) Spore forming species of bacteria;

(2) Spore forming species of fungi;

(3) Mycorrhizal organisms including Laccaria bicolor, Glomusintraradices, and Amanita species;

(4) Actinomyces species and strains thereof, including Streptomyceslydicus, Streptomyces griseoviridis, Streptomyces griseoviridis K61(Mycostop; AgBio development), and Streptomyces microflavus AQ 6121;

(5) Bacillus species and strains thereof, including: Bacillusitcheniformis; Bacillus megaterium; Bacillus pumilus, Bacillusamyloliquefaciens, Bacillus licheniformis; Bacillus oleronius; Bacillusmegaterium; Bacillus mojavensis; Bacillus pumilus; Bacillus subtilis;Bacillus circulans; Bacillus globisporus; Bacillus firmus, Bacillusthuringiensis, Bacillus cereus, Bacillus amyloliquefaciens strain D747(Double Nickel; Certis), Bacillus firmus strain I-1582 (Votivo andNortica; Bayer), Bacillus licheniformis, Bacillus licheniformis strainSB3086 (EcoGuard; Novozymes), Bacillus pumilus strain GB34 (YieldShield;Bayer), QST2808 (Sonata; Bayer), Bacillus subtilis strains GB03 (Kodiak;Bayer), MBI 600 (Subtilex; Becker Underwood) & QST 713 (Serenade;Bayer), Bacillus subtilis strain GB122 plus Bacillus amyloliquefaciensstrain GB99 (BioYield; Bayer), Bacillus pumilus strain BU F-33, Bacillusthuringiensis galleriae strain SDS-502, Bacillus thuringiensis kurstaki,VBTS 2546, Bacillus cereus BP01, Bacillus subtilis strain EB120,Bacillus subtilis strain J-P13, Bacillus subtilis FB17, Bacillussubtilis strains QST30002 and QST3004 (NRRL B-50421 and NRRLB-50455),Bacillus subtilis strains QST30002 and QST3004 (NRRL B-50421 andNRRLB-50455) sandpaper mutants, Bacillus thuringiensis subsp kurstakistrain VBTS 2477 quadruple enterotoxindeficient mutants, Bacillussimplex strains 03WN13, 03WN23 and 03WN25, Bacillus subtilis strain QST713, Bacillus mycoides isolate BmJ NRRL B-30890, Bacillus subtilisstrain DSM 17231 and B licheniformis strain DSM17236, Bacillusaryabhattai, B. flexus, B. nealsonii, Bacillus sphaericus, Bacillusmegaterium, B. vallismortis, Bacillus amyloliquefaciens (ATCC 23842),Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642),Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792),Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), andBacillus pumilus (NRS-272);

(6) Species of “Plant Growth Promoting Rhizobacteria” (PGPRs) andstrains thereof, including species reported to be capable of nitrogenfixation, for example Gluconacetobacter species (e.g. Gluconacetobacterdiazotrophicus a.k.a. Acetobacter diazotrophicus), Spirillum species(e.g. Spirillum lipoferum), Azospirillum species, Herbaspirillumseropedicae, Azoarcus species, Azotobacter species, Burkholderiaspecies, Burkholderia sp. A396, and Paenibacillus polymyxa;

(7) N-fixing bacterial species and strains thereof, including Rhizobiumspecies (e.g. Bradyrhizobium species such as Bradyrhizobium japonicum,and Rhizobium meliloti);

(8) Microbial species and strains thereof that are known to improvenutrient use efficiency, including Penicillium species (e.g. Penicilliumbilaii, Penicillium bilaji), and Mesorhizobium cicero;

(9) Microbial species and strains thereof that are known to haveinsecticidal or insect repellent effects including Telenomus podisi,Baculovirus anticarsia; Trichogramma pretiosum, Trichogramma galloi,Chromobacterium subtsugae, Trichoderma fertile JM41R, Beauveriabassiana, Beauveria bassiana strain NRRL 30976, Beauveria bassianastrain ATP02, DSM 24665, Paecilomyces fumosoroseus, Trichodermaharzianum, Verticillium lecanii, Isaria fumosorosea CCM 8367(CCEFO.011.PFR), Lecanicillium muscarium, Streptomyces microflavus, andMuscodor albus;

(10) Microbial species and strains thereof that are known to havenematicidal effects e.g. Myrothecium verrucaria, Pasteuria species andstrains thereof including Pasteuria nishizawae, Pasteuria Pasteuriareneformis strain Pr-3, Paecilomyces lilacinus, Chromobacteriumsubtsugae, Pasteuria strain ATCC SD-5832, Metarhizium species, andFlavobacterium species;

(11) Microbial species and strains thereof that are known to haveantifungal, antimicrobial and/or plant growth promoting effects e.g.Gliocladium species, Pseudomonas species (e.g. Pseudomonas fluorescens,Pseudomonas fluorescens D7, P. putida and P. chlororaphis), Pseudomonasfluorescens strain NRRL B-21133, NRRL B-21053 or NRRL B-21102,Pseudomonas fluorescens VP5, Pseudomonas synxantha, Pseudomonasdiazotrophicus, Enterobacter cloacae strain NRRL B-21050, Trichodermaspecies, Trichoderma virens, Trichoderma atroviride strains,Coniothyrium minitans, Gliocladium species, Gliocladium virens,Gliocladium roseum strain 321U, Trichoderma harzianum species,Trichoderma harzianum Rifai, Clonostachys rosea strain 88-710,Pseudomonas rhodesiae FERM BP-10912, Serratia plymuthica CCGG2742,Cryptococcus lavescens strain OH 182.9, Serratia plymuthica,Cladosporium cladosporioides, Mitsuaria species, Coprinus curtus,Virgibacillus halophilus, Saccharomyces species, Metschnikoviafruticola, Candida oleophila, Acremonium species, Pseudozyma aphidis,Pythium oligandrum, Phoma spp strain I-4278, Achromobacter species,Geomyces species, Pseudomonas azotoformans, strain F30A, Brevibacillusparabrevis strain No 4; non-toxigenic Aspergillus strains NRRL 50427,NRRL 50428, NRRL 50429, NRRL 50430 and NRRL 50431, Sphaerodesmycoparasitica strains IDAC 301008-01, -02, or -03, Muscodor albusstrain NRRL 30547 or NRRL30548, Serratia plymuthica CCGG2742,Pseudomonas koreensis strain 101L21, P lini strain 131L01, Pantoeaagglomerans strain 101L31, Streptomyces scopuliridis strain RB72,Acremonium spp endophytes, Streptomyces spp BG76 strain, Paracoccuskondratievae, Enterobacter cloacae, Cryptococcus flavescens,Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapior Lactobacillus zeae, Paenibacillus polymyxa, Serratia plymuthica,Phoma species, Pythium oligandrum, Mycosphaerella species, andVariovorax species;

(12) Bacterial species and strains thereof from the group termedPink-Pigmented Facultative Methylotrophs (PPFMs) includingMethylobacterium species; and

(13) Microbial species and strains thereof that are known to haveherbicidal effect e.g., Pyrenophora semeniperda;

wherein the urease inhibitor is selected from the group consisting ofN-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphorictriamide, thiophosphoryl triamide, phenyl phosphorodiamidate,N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide,phosphoric triamide, hydroquinone, p-benzoquinone,hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines,thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, derivatives thereof, and anycombination thereof; and

wherein the nitrification inhibitor is selected from the groupconsisting of 2-chloro-6-trichloromethylpyridine,5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, dicyandiamide,2-amino-4-chloro-6-methyl-pyrimidine, 1,3-benzothiazole-2-thiol,4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine,3,4-dimethylpyrazole phosphate,2,4-diamino-6-trichloromethyl-5-triazine, polyetherionophores,4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, potassium azide,carbon bisulfide, sodium trithiocarbonate, ammonium dithiocarbamate,2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate,N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammoniumthiosulfate, 1-hydroxypyrazole, 3-methylpyrazole-1-carboxamide,3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77Nitrification Inhibitor (CAS Registration No. 1373256-33-7), derivativesthereof, and any combination thereof.

In one embodiment, the present disclosure provides a solid carrier-basedformulation for any microbial inoculant composition of the presentdisclosure, wherein the solid carrier is selected from mineral earths,e.g. silicates, silica gels, talc, kaolins, limestone, lime, chalk,clays, dolomite, diatomaceous earth, bentonite, montmorillonites;inorganic salts, e.g. aluminum sulfate, calcium sulfate, copper sulfate,iron sulfate, magnesium sulfate, silicon sulfate, magnesium oxide;polysaccharides, e.g. cellulose, starch; fertilizers, e.g., ammoniumsulfate, ammonium phosphate, ammonium nitrate; products of vegetableorigin, e.g. cereal meal, tree bark meal, wood meal, nutshell meal;grain flours suitable for the use in the present disclosure, e.g. floursfrom corn, rice, wheat, barley, sorghum, millet, oat, triticale, rye,buckwheat, fonio and quinoa, and mixtures thereof,

In one embodiment, the present disclosure provides a solvent-basedformulation for any agricultural microbial inoculant composition of thepresent disclosure, wherein the solvent is selected from alkanolaminessuch as triethanolamine, diethanolamine, monoethanolamine;alkyldiethanolamines, dialkylmonoethanolamines, wherein the alkyl groupis C₁-C₂₄ branched or unbranched alkyl chain; dimethylsulfoxide (DMSO);alkylsulfones such as sulfolane(2,3,4,5-tetrahydrothiophene-1,1-dioxide); alkyl amides such asN-methylpyrrolidone, N-ethylpyrrolidone, or dimethylformamide;monoalcohols such as methanol, ethanol, propanol, isopropanol, or benzylalcohol; glycols such as ethylene glycol, propylene glycol, diethyleneglycol, or dipropylene glycol; glycol derivatives and protected glycolssuch as triethylene glycol monobutyl ether; glycerol and glycerolderivatives (trialcohols) including protected glycerols such asisopropylidine glycerol; dibasic esters and derivatives thereof;alkylene carbonates such as ethylene carbonate or propylene carbonate;monobasic esters such as ethyl lactate or ethyl acetate; polymers ofcarboxylic acids such as maleic acid, oleic acid, itaconic acid, acrylicacid, or methacrylic acid; monoalkyl glycol ethers and dialkyl glycolethers; glycol esters; surfactants such as alkylbenzenesulfonates,lignin sulfonates, alkylphenol ethoxylates, or polyethoxylated amines.

In one embodiment, the present disclosure provides an encapsulatedformulation for any agricultural microbial inoculant composition of thepresent disclosure. In the soil environment, inoculated microbialspecies can find survival difficult among naturally occurring competitorand predator organisms. To aid in survival of microorganisms present inmicrobial inoculants and fertilizer compositions of the presentdisclosure upon application in the environment, one or more of themicrobial species strains may be encapsulated in, for example, asuitable polymeric matrix. In one example, encapsulation may comprisealginate beads such as has been described by Young et al, 2006,Encapsulation of plant growth-promoting bacteria in alginate beadsenriched with humid acid, Biotechnology and Bioengineering 95:76-83.Those skilled in the art will appreciate that any suitable encapsulationmaterial or matrix may be used. Encapsulation may be achieved usingmethods and techniques known to those skilled in the art. Encapsulatedmicroorganisms can include nutrients or other components of theinoculant or fertilizer composition in addition to the microorganisms.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof; and

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. a urease inhibitor selected from the group consisting ofN-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphorictriamide, thiophosphoryl triamide, phenyl phosphorodiamidate,N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide,phosphoric triamide, hydroquinone, p-benzoquinone,hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines,thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. a nitrification inhibitor selected from the group consisting of2-chloro-6-trichloromethylpyridine,5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, dicyandiamide,2-amino-4-chloro-6-methyl-pyrimidine, 1,3-benzothiazole-2-thiol,4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine,3,4-dimethylpyrazole phosphate,2,4-diamino-6-trichloromethyl-5-triazine, poly etherionophores,4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, potassium azide,carbon bisulfide, sodium trithiocarbonate, ammonium dithiocarbamate,2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate,N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammoniumthiosulfate, 1-hydroxypyrazole, 3-methylpyrazole-1-carboxamide,3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77Nitrification Inhibitor (CAS Registration No. 1373256-33-7), and anycombination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises a urease inhibitor and at least oneorganic solvent, wherein said urease inhibitor is selected from thegroup consisted of N-(n-butyl)thiophosphoric triamide (NBPT),N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenylphosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexylthiophosphoric triamide, phosphoric triamide, hydroquinone,p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines,thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof; andwherein said organic solvent is selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

Part B, wherein Part B comprises at least one Bacillus species selectedfrom the group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises at least one organic solvent selectedfrom the group consisting of propylene glycol (PG),N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether,glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and

Part B, wherein Part B comprises at least one Bacillus species selectedfrom the group consisting of Bacillus amyloliquefaciens, Bacilluslicheniformis, Bacillus thuringiensis, Bacillus pumilus, and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

In one embodiment, the at least one agriculturally beneficial Bacillusspecies in an agricultural microbial inoculant composition or a kit isselected from the group consisting of Bacillus licheniformis, Bacillusthuringiensis, and any combination thereof.

In one embodiment, the agriculturally beneficial Bacillus species in anagricultural microbial inoculant composition or a kit comprises Bacillusstrain selected from the group consisting of Bacillus amyloliquefaciens(ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacilluslicheniformis (B-642), Bacillus licheniformis (B-14368), Bacillusthuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillusthuringiensis (HD-1), Bacillus pumilus (NRS-272), and any combinationthereof.

In one embodiment, the urease inhibitor in an agricultural microbialinoculant composition or a kit, where present, isN-(n-butyl)thiophosphoric triamide (NBPT).

In one embodiment, the nitrification inhibitor in an agriculturalmicrobial inoculant composition or a kit, where present, isdicyandiamide, G77 Nitrification Inhibitor (CAS Registration No.1373256-33-7), or a combination thereof.

In one embodiment, the solvent in an agricultural microbial inoculantcomposition or a kit comprises propylene glycol andN-methyl-2-pyrrolidone.

In one embodiment, the solvent in an agricultural microbial inoculantcomposition or a kit comprises propylene glycol, N-methyl-2-pyrrolidone,and triethylene glycol monobutyl ether.

In one embodiment, an agricultural microbial inoculant compositionfurther comprises water, glycerol or a combination thereof.

In one embodiment, Part B of an agricultural microbial inoculant kitfurther comprises water, glycerol or a combination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus licheniformis, Bacillus thuringiensis,and any combination thereof; and

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus species selected fromthe group consisting of Bacillus licheniformis, Bacillus thuringiensis,and any combination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. N-(n-butyl)thiophosphoric triamide (NBPT).

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises N-(n-butyl)thiophosphoric triamide(NBPT) and at least one organic solvent selected from the groupconsisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP),triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO),and any combination thereof; and

Part B, wherein Part B comprises at least one Bacillus species selectedfrom the group consisting of Bacillus licheniformis, Bacillusthuringiensis, and any combination thereof,

wherein each Part A and Part B is contained in a separate container.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus strain selected fromthe group consisting of Bacillus amyloliquefaciens (ATCC 23842),Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642),Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792),Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacilluspumilus (NRS-272), and any combination thereof; and

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus strain selected fromthe group consisting of Bacillus amyloliquefaciens (ATCC 23842),Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642),Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792),Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacilluspumilus (NRS-272), and any combination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. N-(n-butyl)thiophosphoric triamide (NBPT).

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant composition comprising:

i. at least one agriculturally beneficial Bacillus strain selected fromthe group consisting of Bacillus amyloliquefaciens (ATCC 23842),Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642),Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792),Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacilluspumilus (NRS-272), and any combination thereof;

ii. at least one organic solvent selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and

iii. dicyandiamide, G77 Nitrification Inhibitor (CAS Registration No.1373256-33-7), or a combination thereof.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises N-(n-butyl)thiophosphoric triamide(NBPT) and at least one organic solvent selected from the groupconsisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP),triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO),and any combination thereof; and

Part B, wherein Part B comprises at least one agriculturally beneficialBacillus strain selected from the group consisting of Bacillusamyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580),Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368),Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17),Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS-272), and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

In one embodiment, the present disclosure provides an agriculturalmicrobial inoculant kit comprising:

Part A, wherein Part A comprises at least one organic solvent selectedfrom the group consisting of propylene glycol (PG),N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether,glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and

Part B, wherein Part B comprises at least one agriculturally beneficialBacillus strain selected from the group consisting of Bacillusamyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580),Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368),Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17),Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS-272), and anycombination thereof,

wherein each Part A and Part B is contained in a separate container.

The weight percentage of a urease inhibitor such as NBPT in anyembodiment of an agricultural microbial inoculant composition or a kitof the present disclosure, where presents, is in the range of 0.02-80%.In one embodiment, the weight percentage is in the range of 0.02-70%. Inone embodiment, the weight percentage is in the range of 0.02-60%. Inone embodiment, the weight percentage is in the range of 0.02-50%. Inone embodiment, the weight percentage is in the range of 0.02-40%. Inone embodiment, the weight percentage is in the range of 0.02-30%. Inone embodiment, the weight percentage is in the range of 0.02-20%. Inone embodiment, the weight percentage is in the range of 0.02-10%. Inone embodiment, the weight percentage is in the range of 0.02-5%. In oneembodiment, the weight percentage is in the range of 5-60%. In oneembodiment, the weight percentage is in the range of 5-50%. In oneembodiment, the weight percentage is in the range of 5-40%. In oneembodiment, the weight percentage is in the range of 5-30%. In oneembodiment, the weight percentage is in the range of 10-60%. In oneembodiment, the weight percentage is in the range of 10-50%. In oneembodiment, the weight percentage is in the range of 10-40%. In oneembodiment, the weight percentage is in the range of 10-30%. In oneembodiment, the weight percentage is in the range of 15-60%. In oneembodiment, the weight percentage is in the range of 15-50%. In oneembodiment, the weight percentage is in the range of 15-40%. In oneembodiment, the weight percentage is in the range of 15-30%. In oneembodiment, the weight percentage is in the range of 30-60%. In oneembodiment, the weight percentage is in the range of 30-50%. In oneembodiment, the weight percentage is in the range of 40-60%. The weightpercentage is based on the entirety of the microbial inoculantcomposition.

The weight percentage of a nitrification inhibitor such as DCD, wherepresent, in any embodiment of an agricultural microbial inoculantcomposition or a kit of the present disclosure is in the range of 1-80%.In one embodiment, the weight percentage is in the range of 1-70%. Inone embodiment, the weight percentage is in the range of 1-60%. In oneembodiment, the weight percentage is in the range of 1-50%. In oneembodiment, the weight percentage is in the range of 1-40%. In oneembodiment, the weight percentage is in the range of 1-30%. In oneembodiment, the weight percentage is in the range of 1-20%. In oneembodiment, the weight percentage is in the range of 1-10%. In oneembodiment, the weight percentage is in the range of 1-5%. In oneembodiment, the weight percentage is in the range of 10-80%. In oneembodiment, the weight percentage is in the range of 10-70%. In oneembodiment, the weight percentage is in the range of 10-60%. In oneembodiment, the weight percentage is in the range of 10-50%. In oneembodiment, the weight percentage is in the range of 10-40%. In oneembodiment, the weight percentage is in the range of 10-30%. In oneembodiment, the weight percentage is in the range of 20-80%. In oneembodiment, the weight percentage is in the range of 20-70%. In oneembodiment, the weight percentage is in the range of 20-60%. In oneembodiment, the weight percentage is in the range of 20-50%. In oneembodiment, the weight percentage is in the range of 20-40%. In oneembodiment, the weight percentage is in the range of 20-30%. The weightpercentage is based on the entirety of the microbial inoculantcomposition.

The weight percentage of an organic solvent such as propylene glycol(PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether,glycerol, dimethyl sulfoxide (DMSO), or any combination thereof in anyembodiment of an agricultural microbial inoculant composition or a kitof the present disclosure is in the range of 20-99%. In one embodiment,the weight percentage is in the range of 20-90%. In one embodiment, theweight percentage is in the range of 20-80%. In one embodiment, theweight percentage is in the range of 20-70%. In one embodiment, theweight percentage is in the range of 20-60%. In one embodiment, theweight percentage is in the range of 20-50%. In one embodiment, theweight percentage is in the range of 30-99%. In one embodiment, theweight percentage is in the range of 30-90%. In one embodiment, theweight percentage is in the range of 30-80%. In one embodiment, theweight percentage is in the range of 30-70%. In one embodiment, theweight percentage is in the range of 30-60%. In one embodiment, theweight percentage is in the range of 30-50%. In one embodiment, theweight percentage is in the range of 40-99%. In one embodiment, theweight percentage is in the range of 40-80%. In one embodiment, theweight percentage is in the range of 40-70%. In one embodiment, theweight percentage is in the range of 40-60%. The weight percentage isbased on the entirety of the microbial inoculant composition.

In one embodiment, an agricultural microbial inoculant composition or akit of the present disclosure comprises propylene glycol with the weightpercentage in the range of 40-70%, N-methyl-2-pyrrolidone with theweight percentage in the range of 15-40%, NBPT with the weightpercentage in the range of 10-30%, and optionally a dye with the weightpercentage in the range of 0.1-5%. The weight percentage is based on theentirety of the microbial inoculant composition.

In one embodiment, an agricultural microbial inoculant composition or ankit of the present disclosure comprises propylene glycol with the weightpercentage in the range of 10-30%, N-methyl-2-pyrrolidone with theweight percentage in the range of 30-60%, NBPT with the weightpercentage in the range of 15-40%, triethylene glycol monobutyl etherwith the weight percentage in the range of 1-5%, and optionally a dyewith the weight percentage in the range of 0-1%. The weight percentageis based on the entirety of the microbial inoculant composition.

In one embodiment, the concentration of the Bacillus species in themicrobial inoculant composition of the present disclosure is at least1.0×10² spores/mL. In one embodiment, the concentration is at least1.0×10³ spores/mL. In one embodiment, the concentration is at least1.0×10⁴ spores/mL. In one embodiment, the concentration is at least1.0×10⁵ spores/mL. In one embodiment, the concentration is at least1.0×10⁶ spores/mL. In one embodiment, the concentration is at least1.0×10⁷ spores/mL. In one embodiment, the concentration is at least1.0×10⁸ spores/mL. In one embodiment, the concentration is at least1.0×10⁹ spores/mL. In one embodiment, the concentration is at least1.0×10¹⁰ spores/mL. In one embodiment, the concentration is at least1.0×10¹¹ spores/mL. In one embodiment, the concentration is at least1.0×10¹² spores/mL. In one embodiment, the concentration is in the rangeof 1.0×10²-1.0×10¹² spores/mL. In one embodiment, the concentration isin the range of 1.0×10³-1.0×10¹² spores/mL. In one embodiment, theconcentration is in the range of 1.0×10⁴-1.0×10¹² spores/mL. In oneembodiment, the concentration is in the range of 1.0×10⁵-1.0×10¹²spores/mL. In one embodiment, the concentration is in the range of1.0×10⁶-1.0×10¹² spores/mL. In one embodiment, the concentration is inthe range of 1.0×10⁷-1.0×10¹² spores/mL. In one embodiment, theconcentration is in the range of 1.0×10⁸-1.0×10¹² spores/mL. In oneembodiment, the concentration is in the range of 1.0×10⁹-1.0×10¹²spores/mL.

In one embodiment of the present disclosure, the agricultural microbialinoculant composition may serve as a fertilizer by itself.

A dye may also be included in the agricultural microbial inoculantcomposition in the present disclosure. Any commonly used dye includingfood dyes may be used to provide visual evidence of the uniformity ofthe microbial inoculant composition. The weight percentage of a dye inthe total microbial inoculant composition is 0-10%. In one embodiment,the weight percentage is 0.1-5%.

Examples of dyes suitable in the present disclosure include but are notlimited to FD&C Blue No. 1, FD&C Blue No. 1, FD&C Green No. 3, FD&CYellow No. 5, FD&C Red No. 3, FD&C Red No. 40, FD&C Yellow No. 6, andAGROTAIN® ULTRA green dye, or a combination thereof.

In another embodiment, the present disclosure provides a fertilizercomposition comprising any agricultural microbial inoculant compositionin any embodiment of the present disclosure, wherein the fertilize canbe a granular fertilizer such as urea granular, a liquid fertilizer suchas urea ammonium nitrate (UAN), an aqueous urea and ammonia nitrateaqueous solution, or anhydrous ammonia (NH₃).

In another embodiment, the present disclosure provides a method ofenhancing a yield trait in a subject plant as compared to the yieldtrait of a reference or control plant, the method comprising contactinga subject plant, plant part, plant seed, or surrounding soil with aneffective amount of an agricultural microbial inoculant composition ofthe present disclosure, wherein the microbial inoculant compositioncomprises:

i. at least one agricultural microbial strain from one or more microbialspecies, and

ii. at least one active agent, wherein the active agent is a ureaseinhibitor, nitrification inhibitor, or a combination thereof,

wherein the agricultural microbial inoculant composition at theeffective amount is effective in enhancing the yield trait in thesubject plant relative to the yield trait in the reference or controlplant when the subject plant is contacted with the effective amount.

In another embodiment, the present disclosure provides a method forenhancing a yield trait in the plant, such as increasing plant growthand/or productivity, wherein the method comprises applying to the plant,plant part, plant seeds or to the soil in which the plant or plant seedsare grown an effective amount of an agricultural microbial inoculantcomposition of any embodiment of the present disclosure.

In another embodiment, the present disclosure provides a method forimproving soil quality, wherein the method comprises applying to soil orto the plants or plant seeds in said soil an effective amount of anagricultural microbial inoculant composition as disclosed in anyembodiment of the present disclosure.

In any embodiment of the disclosure, the concentrations of eachagricultural microbial strain to be added to microbial inoculants andfertilizer compositions as disclosed herein will depend on a variety offactors including the identity and number of individual strainsemployed, the plant species being treated, the nature and condition ofthe soil to be treated, the exact nature of the microbial inoculant orfertilizer composition to be applied, the type and form of active agent,the form in which the inoculant or fertilizer is applied and the meansby which it is applied, and the stage of the plant growing season duringwhich application takes place. For any given case, appropriateconcentrations should be effective in enhancing the yield trait in thepresence of the active agent, and may be determined by one of ordinaryskill in the art using only routine experimentation. By way of exampleonly, the concentration of each strain present in the inoculant orfertilizer composition may be from about 1.0×10² colony forming units(CFU)/mL to about 5.0×10¹² CFU/mL per acre, from about 1.0×10² CFU/mL toabout 5.0×10¹⁰ CFU/mL per acre, from about 1.0×10² CFU/mL to about5.0×10⁸ CFU/mL per acre, from about 1.0×10² CFU/mL to about 5.0×10⁶CFU/mL per acre, or from about 1.0×10² CFU/mL to about 5.0×10⁴ CFU/mLper acre.

In one embodiment of the present disclosure, a microbial food sourcesuch as kelp or glycerol may be included in any embodiment of thepresent disclosure.

The term “microbial species” refers to either naturally occurring orspecifically developed variants or mutants of microbial species such asbacteria and fungi as disclosed herein. Variants or mutants may or maynot have the same identifying biological characteristics of the specificstrains exemplified herein, provided they share similar advantageousproperties in terms of promoting plant growth and providing nutrientsfor plant growth in the soil. Variants of certain microbial strains mayinclude but not limited to those developed by gene integrationtechniques such as those mediated by insertional elements or transposonsor by homologous recombination, other recombinant DNA techniques formodifying, inserting, deleting, activating or silencing genes,intraspecific protoplast fusion, mutagenesis by irradiation withultraviolet light or X-rays, or by treatment with a chemical mutagensuch as nitrosoguanidine, methylmethane sulfonate, nitrogen mustard andthe like, and bacteriophage-mediated transduction. Suitable andapplicable methods are well known in the art and are described, forexample, in J. H. Miller, Experiments in Molecular Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller,A Short Course in Bacterial Genetics, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1992); and J. Sambrook, D. Russell,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001).

The term “plant productivity” or “yield trait” as used herein refers toany aspect of growth or development of a plant that is a reason forwhich the plant is grown. Thus, for purposes of the present disclosure,improved or increased “plant productivity” or “enhanced yield trait”refers broadly to improvements in biomass or yield of leaves, stems,grain, fruit, vegetables, flowers, or other plant parts harvested orused for various purposes, and improvements in growth of plant parts,including stems, leaves and roots.

The term “improving soil quality” refers to the increasing the amountand/or availability of nutrients required by, or beneficial to plants,for growth. For example only, such nutrients include nitrogen,phosphorous, potassium, copper, zinc, boron and molybdenum. Alsoencompassed by the term “improving soil quality” is reducing orminimizing the amount of an element that may be detrimental to plantgrowth or development such as, for example iron and manganese. Thus,improving soil quality by use of microbial inoculants and fertilizercompositions of the present disclosure thereby assists and promotes thegrowth of plants in the soil.

The term “effective amount” refers to an amount of microbial inoculantor fertilizer composition applied to a given area of soil or vegetationthat is sufficient to promote one or more beneficial or desiredoutcomes, for example, in terms of plant growth rates, crop yields, ornutrient availability in the soil. An “effective amount” can be providedin one or more administrations. The exact amount required will varydepending on factors such as the identity and number of individualstrains employed, the plant species being treated, the nature andcondition of the soil to be treated, the exact nature of the microbialinoculant or fertilizer composition to be applied, the form in which theinoculant or fertilizer is applied and the means by which it is applied,and the stage of the plant growing season during which application takesplace. For any given case, an appropriate “effective amount” may bedetermined by one with ordinary skill in the art using only routineexperimentation.

The term “viability” in the present disclosure refers to the capabilityof repeated division of a microbial cell on an agar surface to produce avisible colony. The temperature for the viability evaluation is about37° C. in the present disclosure. The viability time is counted from themoment a freshly prepared microbial culture in a nutrient broth is addedto an organic solvent or a mixture of more than one organic solvent.After the microbial culture in a nutrient broth is added to the organicsolvent or the mixture of more than one solvent, a small amount ofmixture is taken out at certain time for incubation at about 37° C. Thetime period between the moment the microbial culture in a nutrient brothis added to an organic solvent and the moment that the mixture is takenout for incubation is defined as the viability time for the viabilityevaluation purpose.

Bacteria Viability Test in Solutions with Organic Solvents

The purpose of the bacteria viability test is to evaluate the viabilityof agriculturally beneficial bacteria species in solutions with organicsolvents.

Both agriculturally beneficial Gram-positive and Gram-negative bacteriaspecies are used for the test.

Agriculturally beneficial bacteria species were obtained from theAmerican Type Culture Collection (ATCC), or the Agricultural ResearchService Culture Collection. The bacteria species were grown onappropriate media, LB broth, nutrient broth, and checked microscopicallywith Gram staining and on Petri plates for purity. The bacteria speciesare pure, i.e., no unusual colonies were observed.

All the samples for testing are prepared by a two-step method.

First, selected agriculturally beneficial bacteria were grown overnightin Luria-Bertani (LB) medium at 37° C. with agitation. Growth wasmeasured with a Bausch and Lomb Spectronic Spectrophotometer at at 600nm (OD₆₀₀) to provide samples with cell optical density at 600 nm(OD₆₀₀) between 1.2 and 1.5. The overnight cultures typically contain1.6×10⁸ to 3.4×10⁸ colony forming units per mL (CFU/mL).

Second, 0.5 mL of the prepared bacteria species sample in LB medium wasadded to an organic solvent or a mixture of organic solvents (4.5 mL).

A sample of 10 μL of the organic solvent solution with bacteria speciesis removed immediately for bacteria viability evaluation (T=0).

The remaining organic solvent solution with the bacteria species isincubated at 37° C. for future test. A volume of 10 μL of sample wastaken out from the incubated organic solvent solution with the bacteriaspecies at T=2 hours, 4 hours, 1 day, 2 days, 5 days, 7 days, 9 days, 12days, 15 days, 18 days and 21 days for bacteria viability evaluation.

Each time the sample (10 μL) that was taken out for viability evaluationwas placed onto agar plates, which was incubated overnight at 37° C.Colonies of bacteria present on the plates indicates that the bacteriahave tolerated the solvent and was therefore viable. If there is nogrowth of colonies of bacteria, the bacteria have demonstrated notolerance in the solvent.

The agriculturally beneficial bacteria that are viable for at least twohours from the moment that the bacteria sample is added to an organicsolvent are considered to be viable in the organic solvent solution ofthe present invention.

Bacillus amyloliquefaciens (ATCC 23842) provided at least two hours ofviability in NMP, and at least 5 days of viability in glycerol.

Bacillus licheniformis (ATCC 14580) provided at least one day ofviability in triethylene glycol monobutyl ether, at least 5 days ofviability in PG, at least 5 days of viability in NMP, at least 21 daysof viability in glycerol, and at least 21 days of viability in DMSO.

Bacillus licheniformis (B-642) and Bacillus licheniformis (B-14368) eachprovided at least 21 days of viability in PG, at least 21 days ofviability in NMP, at least 21 days of viability in triethylene glycolmonobutyl ether, at least 21 days of viability in glycerol, and at least21 days of viability in DMSO.

Bacillus thuringiensis (ATCC 10792) provided at least 2 hours ofviability in PG, at least 2 hours of viability in NMP, at least 2 hoursof viability in triethylene glycol monobutyl ether, at least 21 days ofviability in glycerol, and at least 21 days of viability in DMSO.

Bacillus thuringiensis (HD-17) and Bacillus thuringiensis (HD-1) eachprovided at least 21 days of viability in PG, at least 21 days ofviability in NMP, at least 21 days of viability in triethylene glycolmonobutyl ether, at least 21 days of viability in glycerol, and at least21 days of viability in DMSO.

Bacillus pumilus (NRS-272) provided at least 21 days of viability in PG,at least 21 days of viability in triethylene glycol monobutyl ether, atleast 21 days of viability in glycerol, and at least 21 days ofviability in DMSO.

Surprisingly, all three selected agriculturally beneficial Gram-negativespecies Pseudomonas fluorescens (ATCC 53958), Pseudomonas putida (ATCC49128), and Pseudomonas chlororaphis (ATCC 55670), which were expectedto have better solvent-tolerance, lost viability almost instantly whenthe prepared bacteria samples with OD₆₀₀ between 1.3 and 1.5 in nutrientbroth were added to all tested organic solvent except glycerol.

The bacteria viability test in solutions with organic solvents in thepresent disclosure demonstrated that all the examples of Bacilluslicheniformis and Bacillus licheniformis provided viability with timerange from at least 2 hours to at least 21 days in a solution with atleast one of the organic solvents PG, NMP, triethylene glycol monobutylether, glycerol and DMSO.

The bacteria viability test in solutions with organic solvents in thepresent disclosure demonstrated that the Bacillus strains Bacillusamyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580),Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368),Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17),Bacillus thuringiensis (HD-1) and Bacillus pumilus (NRS-272) eachprovided viability with time range from at least 2 hours to at least 21days in a solution with at least one of the organic solvents PG, NMP,triethylene glycol monobutyl ether, glycerol and DMSO.

Examples

TABLE 1 Examples with viability of at least 2 hours in at least one ofthe organic solvents PG, NMP, triethylene glycol monobutyl ether,glycerol or DMSO Example No. Bacillus Species Bacillus Strains Solvent 1amyloliquefaciens ATCC 23842 NMP 2 amyloliquefaciens ATCC 23842 Glycerol3 licheniformis ATCC 14580 Triethylene glycol monobutyl ether 4licheniformis ATCC 14580 PG 5 licheniformis ATCC 14580 NMP 6licheniformis ATCC 14580 glycerol 7 licheniformis ATCC 14580 DMSO 8licheniformis B-642 PG 9 licheniformis B-642 NMP 10 licheniformis B-642Triethylene glycol monobutyl ether 11 licheniformis B-642 Glycerol 12licheniformis B-642 DMSO 13 licheniformis B-14368 PG 14 licheniformisB-14368 NMP 15 licheniformis B-14368 Triethylene glycol monobutyl ether16 licheniformis B-14368 Glycerol 17 licheniformis B-14368 DMSO 18thuringiensis ATCC 10792 PG 19 thuringiensis ATCC 10792 NMP 20thuringiensis ATCC 10792 Triethylene glycol monobutyl ether 21thuringiensis ATCC 10792 Glycerol 22 thuringiensis ATCC 10792 DMSO 23thuringiensis HD-17 PG 24 thuringiensis HD-17 NMP 25 thuringiensis HD-17Triethylene glycol monobutyl ether 26 thuringiensis HD-17 Glycerol 27thuringiensis HD-17 DMSO 28 thuringiensis HD-1 PG 29 thuringiensis HD-1NMP 30 thuringiensis HD-1 Triethylene glycol monobutyl ether 31thuringiensis HD-1 Glycerol 32 thuringiensis HD-1 DMSO 33 pumilisNRS-272 PG 34 pumilis NRS-272 Triethylene glycol monobutyl ether 35pumilis NRS-272 Glycerol 36 pumilis NRS-272 DMSO

What is claimed is:
 1. A carrier-based agricultural microbial inoculantcomposition comprising: i. at least one agriculturally beneficialBacillus species selected from the group consisting of Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis,Bacillus pumilus, and any combination thereof; ii. at least one organicsolvent selected from the group consisting of propylene glycol (PG),N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether,glycerol, dimethyl sulfoxide (DMSO), and any combination thereof,wherein the at least one organic solvent is present in an amount of20-99 weight percent, based on the total weight of the microbialinoculant composition; and iii a urease inhibitor selected from thegroup consisting of N-(n-butyl)thiophosphoric triamide (NBPT),N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenylphosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexylthiophosphoric triamide, phosphoric triamide, hydroquinone,p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines,thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof. 2.The agricultural microbial inoculant composition of claim 1, wherein theagriculturally beneficial Bacillus species is selected from the groupconsisting of Bacillus licheniformis, Bacillus thuringiensis, and anycombination thereof.
 3. The agricultural microbial inoculant compositionof claim 1, wherein the agriculturally beneficial Bacillus species isselected from the group of strains consisting of Bacillusamyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580),Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368),Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17),Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS-272), and anycombination thereof.
 4. The agricultural microbial inoculant compositionof claim 1, wherein the urease inhibitor is N-(n-butyl)thiophosphorictriamide (NBPT).
 5. The agricultural microbial inoculant composition ofclaim 1, further comprising a nitrification inhibitor selected from thegroup consisting of 2-chloro-6-trichloromethylpyridine,5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, dicyandiamide,2-amino-4-chloro-6-methyl-pyrimidine, 1,3-benzothiazole-2-thiol,4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine,3,4-dimethylpyrazole phosphate,2,4-diamino-6-trichloromethyl-5-triazine, poly etherionophores,4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, potassium azide,carbon bisulfide, sodium trithiocarbonate, ammonium dithiocarbamate,2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate,N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammoniumthiosulfate, 1-hydroxypyrazole, 3-methylpyrazole-1-carboxamide,3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77Nitrification Inhibitor (CAS Registration No. 1373256-33-7), and anycombination thereof.
 6. The agricultural microbial inoculant compositionof claim 5, wherein the nitrification inhibitor is dicyandiamide, G77Nitrification Inhibitor (CAS Registration No. 1373256-33-7) or acombination thereof.
 7. The agricultural microbial inoculant compositionof claim 1, wherein the weight percentage of the urease inhibitor is inthe range of 0.02-80% based on the total weight of the agriculturalmicrobial inoculant composition.
 8. The agricultural microbial inoculantcomposition of claim 7, wherein the weight percentage of the ureaseinhibitor is in the range of 10-60%.
 9. The agricultural microbialinoculant composition of claim 1, wherein the agriculturally beneficialBacillus species has at least 2 hours of viability from the time whensaid agriculturally beneficial Bacillus species contacts said organicsolvent.
 10. The agricultural microbial inoculant composition of claim1, wherein the agriculturally beneficial Bacillus species concentrationis in the range of 1.0×10²-1.0×10¹² spores/mL.
 11. The agriculturalmicrobial inoculant composition of claim 1, further comprising a dye.12. The agricultural microbial inoculant composition of claim 1,comprising propylene glycol and N-methyl-2-pyrrolidone.
 13. Theagricultural microbial inoculant composition of claim 12, wherein theweight percentage of propylene glycol is in the range of 40-70% based onthe total weight of the agricultural microbial inoculant composition,the weight percentage of N-methyl-2-pyrrolidone is in the range of15-40% based on the total weight of the agricultural microbial inoculantcomposition, the urease inhibitor is NBPT, and the weight percentage ofNBPT is in the range of 10-30% based on the total weight of themicrobial inoculant composition.
 14. The agricultural microbialinoculant composition of claim 1 comprising propylene glycol,N-methyl-2-pyrrolidone, and triethylene glycol monobutyl ether.
 15. Theagricultural microbial inoculant composition of claim 14, wherein theweight percentage of propylene glycol is in the range of 10-30% based onthe total weight of the agricultural microbial inoculant composition,the weight percentage of N-methyl-2-pyrrolidone is in the range of30-60% based on the total weight of the agricultural microbial inoculantcomposition, the weight percentage of triethylene glycol monobutyl etheris in the range of 1-5% based on the total weight of the agriculturalmicrobial inoculant composition, the urease inhibitor is NBPT, and theweight percentage of NBPT is in the range of 15-40% based on the totalweight of the agricultural microbial inoculant composition.
 16. Afertilizer composition comprising the agricultural microbial inoculantcomposition according to claim
 1. 17. A method for promoting plantgrowth, plant productivity and/or soil quality, wherein the methodcomprises applying an effective amount of the agricultural microbialinoculant composition according to claim 1 to a plant, plant part, plantseed or soil.
 18. An agricultural microbial inoculant kit comprising:Part A, wherein Part A comprises a urease inhibitor and at least oneorganic solvent, wherein said urease inhibitor is selected from thegroup consisting of N-(n-butyl)thiophosphoric triamide (NBPT),N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenylphosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexylthiophosphoric triamide, phosphoric triamide, hydroquinone,p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines,thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone,N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide,N-(2-nitrophenyl)phosphoric triamide, and any combination thereof; andwherein said organic solvent is selected from the group consisting ofpropylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycolmonobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and anycombination thereof; and Part B, wherein Part B comprises at least oneBacillus species selected from the group consisting of Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis,Bacillus pumilus, and any combination thereof, wherein each of Part Aand Part B is contained in a separate container, and wherein the atleast one organic solvent is present in an amount of 20-99 weightpercent, based on the combined weight of Part A and Part B.
 19. Theagricultural microbial inoculant kit of claim 18, wherein Part Bcomprises at least one Bacillus species selected from the groupconsisting of Bacillus licheniformis, Bacillus thuringiensis, and anycombination thereof.
 20. The agricultural microbial inoculant kit ofclaim 18, wherein Part B comprises at least one agriculturallybeneficial Bacillus strain selected from the group consisting ofBacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC14580), Bacillus licheniformis (B-642), Bacillus licheniformis(B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis(HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS-272), andany combination thereof.
 21. The agricultural microbial inoculant kit ofclaim 18, wherein the urease inhibitor is N-(n-butyl)thiophosphorictriamide (NBPT).
 22. The agricultural microbial inoculant kit of claim21, wherein Part A comprises propylene glycol andN-methyl-2-pyrrolidone.
 23. The agricultural microbial inoculant kit ofclaim 22, wherein the weight percentage of propylene glycol is in therange of 40-70% based on the total weight of Part A, the weightpercentage of N-methyl-2-pyrrolidone is in the range of 15-40% based onthe total weight of Part A, and the weight percentage of NBPT is in therange of 10-30% based on the total weight of Part A.
 24. Theagricultural microbial inoculant kit of claim 21, wherein Part Acomprises propylene glycol, N-methyl-2-pyrrolidone, and triethyleneglycol monobutyl ether.
 25. The agricultural microbial inoculant kit ofclaim 24, wherein the weight percentage of propylene glycol is in therange of 10-30% based on the total weight of Part A, the weightpercentage of N-methyl-2-pyrrolidone is in the range of 30-60% based onthe total weight of Part A, the weight percentage of triethylene glycolmonobutyl ether is in the range of 1-5% based on the total weight ofPart A, and the weight percentage of NBPT is in the range of 15-40%based on the total weight of Part A.
 26. The agricultural microbialinoculant kit of claim 18, wherein the agriculturally beneficialBacillus species has at least 2 hours of viability from the time whensaid agriculturally beneficial Bacillus species in Part A contacts saidorganic solvent in Part B.
 27. The agricultural microbial inoculant kitof claim 18, wherein the weight percentage of the urease inhibitor is inthe range of 0.02-80% based on the total weight of Part A.
 28. Theagricultural microbial inoculant kit of claim 18, wherein theagriculturally beneficial Bacillus species concentration is in the rangeof 1.0×10² to 1.0×10¹² spores/mL after Part A is mixed with Part B.